Project Summary Cigarette smoking causes 480,000 deaths annually, making nicotine about 10 times more lethal than opioids. In addition, smoking-related illnesses in the United States cost more than $300 billion each year. Both genetic factors and social environment have a strong in?uence on smoking behavior. At least 26 human genome-wide association studies (GWAS) on smoking have been conducted to date. Only 11 loci, each accounting for 0.2?0.99% of the variances of a few self-reported phenotypes have been replicated. We have developed a model of nicotine self-administration in adolescent rats that captures the role of social learning in promoting nicotine intake. This operant licking model delivers intravenous nicotine with a contingent oral ?avor (i.e., taste and odor) cue. We found social learning facilitated the extinction of conditioned nicotine aversion and promoted nicotine intake. In the prior funding period, we have almost ?nished phenotyping 1,600 adolescent heterogenous stock male and female rats using this model. We also measured several social, novelty-seeking and anxiety-like behaviors in these rats. Our regression analysis showed that social and emotional-like behaviors explain approximately 30% of the variance in nicotine intake. We also sequenced the transcriptome of 440 samples from nave rats. Our genetic analysis has identi?ed many quantitative trait loci (QTL) for both behavior and gene expression phenotypes. Human GWAS has shown that increasing sample size exponentially increases the number of signi?cant associations. Similarly, we have completed a GWAS of body weight and related traits using almost 3,200 rats. By examining what we would have found with only 1,600 rats, we show the increase in QTL from 1,600 to 3,200 is exponential rather than linear. Therefore, in this renewal application, we are proposing to extend our study by phenotyping an additional 1,600 rats, which will bring our ?nal sample size to 3,200. We anticipate the combined study will identify genes involved in different aspects of nicotine addiction, such as the rewarding and aversive effects of nicotine, progression of nicotine intake, and relapse, among many others. We plan to maintain the experimental design from the last funding period, because it worked well, and to assure that the full cohort of 3,200 rat is as homogeneous as possible. However, we will add a new social interaction test, where we will analyze the social behaviors of two freely moving rats using Yorodent, an arti?cial intelligence-based analysis method developed in our lab. In Aim 1, we will phenotype adolescent heterogeneous rats. Breeders will be obtained from Core B (HS Breeding Core), which we will use to generate 400 adolescent rats per year in years 1-4. These rats will ?rst be phenotyped for their social, novelty-seeking and anxiety-like behaviors. They then will be implanted with a jugular catheter. Nicotine IVSA will start on postnatal day 38. In Aim 2, We will analyze the relationships between behavioral traits using regression and genetic correlations. We will also perform a phenome-wide associations study to identify pleiotropic effects of genetic variants identi?ed in this project. In Aim 3, we will obtain brain tissues that are anatomically precise from nave rats to expand our transcriptome database. These data will provide mechanistic insights for behavior associations obtained from Projects 1?3 and be used by Project 4 for network analysis.